Mapping of intra locus duplications and introgressed DNA:: aids to map-based cloning of genes from complex genomes illustrated by physical analysis of the Rx locus in tetraploid potato

We describe here novel approaches to high-resolution mapping of repeated and introgressed DNA which may prove generally useful in map-based cloning from complex genomes, These approaches were developed in order to clone the Rs locus in potato. First, we prepared a BAC library from a tetraploid plant carrying Rx in the duplex condition (Rx, Rx, rx, rx). BAC clones were then isolated with Close markers on either side of Rx. However these clones did not extend across Rx: in the cloned DNA the closest markers to Rx were separated by single recombination events on either side of Rx. To bridge the gap we exploited the finding that the BAC clones on the right side of Rx contained resistance-gene homologues, Anticipating that there would be duplicated copies of these resistance gene homologues in the vicinity of Rx, we used low-stringency PCR conditions to identify additional markers. One of these markers was completely linked to Rx in our mapping population and was used to isolate a BAC (BAC77) that had not been previously identified by screening with Rx-flanking markers. Based on two criteria it was concluded that BAC77 spans Rx. There was a chromosomal recombination in one plant of our mapping population that separated the BAC77 right end from Rx, On the other side of Rx it was found that the BAC77 left end was outside the region of DNA carrying Rx that had been introgressed into potato cv Amaryl from a Solanum tuberosum ssp. andigena accession CPC1673.